Bacteria and bacteriophage detection using immobilized enzyme substrates

ABSTRACT

Methods of detecting bacteria including the use of an immobilized enzyme substrate, and the immobilized enzyme substrate.

BACKGROUND OF THE INVENTION

Detection of bacteria is important in a variety of industries, includingthe food and beverage industry. For example, the need to screen food andwater for pathogenic bacteria is crucial to ensuring consumer safety.The determination of levels of certain families of bacteria is acommonly used approach to estimating the shelf life and microbialacceptability of food products and hygienic status of the processingequipment and raw materials used in their manufacture. The diagnosis ofmicrobial infections also relies on the detection of the causativeorganism(s).

There are many methods known for detecting bacteria. For example,bacteriophage, which are viruses that infect bacteria, may be employed.The presence of the bacteriophage, the infected bacteria, or the lackthereof, may be detected. Typically, a target bacteria is detected byinfecting the bacteria with a bacteriophage (BP) specific to thebacteria, inactivating the excess BP, and then manipulating theBP-infected bacteria in some manner to detect the presence or absence ofthe BP as an indirect indication of whether or not the sample originallycontained the target bacteria. Bacterial “helper cells” can be used toamplify the number of BP-infected bacteria and thereby enhance, e.g.,make more rapid, the assay method. A common detection method in thefinal stages of such an assay is to incubate the bacterial helper cellswith the BP-infected bacteria and either observe changes in solutionturbidity or, alternatively, observe BP plaque formation on anappropriate growth medium.

For example, U.S. patent application Ser. No. 09/434,586 (Wicks et al.)describes devices and methods for the detection of bacteria in a sample.Briefly, a sample containing suspect (target) bacteria is infected witha BP specific to the suspect bacteria, the excess BP is inactivated withan antiviral agent, and the BP-infected bacteria are added to bacterialhelper cells to amplify the BP and to produce a signal that can bedetected visually or with an instrument. For example, the BP can bedetected by incubating the helper cells on agar and counting the numberof BP plaques that are formed.

It would be very useful in such assay methods to employ enzymesubstrates (ES) as indicators for detecting the presence of BP (and,thus, indirectly the presence or absence of target bacteria). UtilizingES indicators could lead to significant advantages over trying toobserve changes in solution turbidity or counting plaque formation. Theuse of ES indicators could lead to more convenient, more rapid, and lessexpensive assay methods. However, the use of traditional soluble ESindicators is generally not possible in such assay methods. The solubleES would undesirably react with enzyme within the intact bacteria cellsof both non-target bacteria and, if used, bacterial helper cells andthereby produce unacceptable levels of background signal.

SUMMARY OF THE INVENTION

The present invention solves the problem of the prior art by utilizingenzyme substrates as indicators that have been bonded (i.e.,immobilized) to an insoluble solid support. The use of an immobilizedenzyme substrate prevents the enzyme substrate from crossing a bacteriacell wall to react with enzyme within intact bacteria cells. As aresult, the enzyme substrate can only react with an enzyme released froma lysed bacteria cell.

Thus, the present invention provides a method of detecting (identifyingand/or quantifying) a target bacteriophage. The method includes:combining bacteria and a sample of interest to form a reaction mixture;incubating the reaction mixture under conditions effective for anytarget bacteriophage present in the sample of interest to lyse thebacteria and release enzyme; adding an immobilized enzyme substrate tothe reaction mixture; and monitoring the reaction mixture for adetectable signal produced from interaction between the immobilizedenzyme substrate and any released enzyme present. Adding the immobilizedenzyme substrate to the reaction mixture can occur before or afterincubating the reaction mixture. This method can involve a qualitativeor quantitative determination of bacteriophage in a sample. For aquantitative determination, the reaction mixture is plated out on anappropriate growth medium, and the areas emitting the detectable signalare counted.

Alternatively, there is provided a method of detecting (identifyingand/or quantifying) target bacteria. The method includes: combiningbacteriophage and a sample of interest to form a reaction mixture;incubating the reaction mixture under conditions effective for thebacteriophage to lyse any target bacteria present in the sample ofinterest and release enzyme; adding an immobilized enzyme substrate tothe reaction mixture; and monitoring the reaction mixture for adetectable signal produced from interaction between the immobilizedenzyme substrate and any released enzyme present. Adding the immobilizedenzyme substrate to the reaction mixture can occur before or afterincubating the reaction mixture. This method can involve a qualitativeor quantitative determination of bacteria in a sample.

In a preferred embodiment, the present invention provides a method ofdetecting target bacteria that involves: combining bacteriophage and asample of interest to form a reaction mixture; allowing thebacteriophage to infect any target bacteria present in the sample ofinterest; adding an antiviral agent to inactivate any extracellularbacteriophage; adding bacterial helper cells to the reaction mixture;adding an immobilized enzyme substrate to the reaction mixture;incubating the reaction mixture under conditions effective for thebacteriophage to lyse any target bacteria present and the bacterialhelper cells and release enzyme; and monitoring the reaction mixture fora detectable signal produced from interaction between the immobilizedenzyme substrate and any released enzyme present. This method ispreferably used for the quantitative determination of bacteria in asample, although it can also involve a qualitative determination.

The present invention also provides an immobilized enzyme substrate thatincludes a porous solid support and an enzyme substrate covalentlybonded thereto.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a method of detecting the presence orabsence of bacteriophage or bacteria using an immobilized enzymesubstrate (i.e., enzyme reactant). Thus, the present invention providesa method that uses enzyme activity for the detection of bacteriophage,which provides an indirect method for the detection of bacteria, or forthe detection of bacteria directly.

The enzyme substrate preferably includes a detectable label that, uponcontact with enzyme present in the sample to be tested, produces achange, for example, in the spectral properties of the enzyme substrateor its reaction products resulting from the enzyme reaction. This changeis used for the determination of the enzyme activity, and hence, thepresence or absence of bacteriophage, and hence bacteria, or bacteriadirectly. Preferably, the change is a spectral change in thefluorescence radiation of the enzyme substrate, although other spectralchanges can be used such as changes in absorption or excitation, forexample.

The enzyme substrate can be immobilized on a variety of solid supports.Preferably, it is a porous support, although other supports can be usedsuch as an optical fiber, as disclosed in U.S. Pat. No. 5,238,809(Wolfbeis). In this latter embodiment, the enzyme substrate is attachedto the end of an optical fiber and a photodetector for subsequent signalevaluation is provided, which will measure the signal, e.g., fluorescentlight, emitted by the enzyme substrate or its reaction products, uponreaction with an enzyme. Suitable photodetectors are photomultipliers,phototransistors and photodiodes. Preferably, the optical fiber is asingle fiber, but it may also be configured as a multi-fiber bundle.

Solid Support

Acceptable supports for use in the present invention can vary widely. Asupport can be porous or nonporous, but is preferably porous. It can becontinuous or noncontinuous, flexible or nonflexible. A support can bemade of a variety of materials including supports made of ceramic,glassy, metallic, organic polymeric materials, or combinations thereof.Such supports can be magnetic, which allows for concentration andintensification of the signal.

Preferred supports include organic polymeric supports, such asparticulate or beaded supports, woven and nonwoven webs (such as fibrouswebs), microporous fibers, microporous membranes, hollow fibers ortubes. Woven and nonwoven webs may have either regular or irregularphysical configurations of surfaces.

Porous materials are particularly desirable because they provide largesurface areas. The porous support can be synthetic or natural, organicor inorganic. Suitable solids with a porous structure having pores of adiameter of at least about 1.0 nanometer (nm) and a pore volume of atleast about 0.1 cubic centimeter/gram (cm³/g). Preferably, the porediameter is at least about 30 nm because larger pores will be lessrestrictive to diffusion. Preferably, the pore volume is at least about0.5 cm³/g for greater potential capacity due to greater surface areasurrounding the pores. Preferred porous supports include particulate orbeaded supports.

For significant advantage, the supports are preferably hydrophilic, andhave high molecular weight (preferably, greater than about 5000, andmore preferably, greater than about 40,000). Preferably, the hydrophilicpolymers are water swellable to allow for greater infiltration ofenzyme. Examples of such supports include cellulose, modifiedcelluloses, agarose, polyvinyl alcohol (PVA), dextrans, amino-modifieddextrans, polyacrylamide, modified guar gums, guar gums, xanthan gums,and locust bean gums.

In order to be useful for the purposes of the invention, the supportincludes a reactive functional group that can be used for coupling tothe enzyme substrate, preferably through a spacer group. Preferably, thereactive functional group is capable of undergoing rapid, direct,covalent coupling with the desired spacer group to form derivatizedsupports. Preferably, the support includes at least one reactivefunctional group, such as a hydroxyl, carboxyl, sulfhydryl, or aminogroup that chemically binds to the enzyme substrate, optionally througha spacer group. Other suitable functional groups includeN-hydroxysuccinimide esters, sulfonyl esters, iodoacetyl groups,aldehydes, imidazolyl carbamates, and cyanogen bromide activatedsupports. Such functional groups can be provided to a support by avariety of known techniques. For example, a glass surface can bederivatized with aminopropyl triethoxysilane in a known manner.

Coupling agents are preferably used in bonding the enzyme substrate to asupport. For example, the coupling agents EDC(1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride) and HOBt(1-hydroxy-benzotriazole hydrate) can aid in covalently bonding acarboxyl group of the enzyme substrate to an amino group of anamino-modified support. The use of such coupling agents is described inAdvanced Organic Chemistry, Jerry March, Wiley InterScience, 4^(th)Edition, 1992, pp 420-421.

Spacer groups can also be used for bonding the enzyme substrate to asupport. Suitable spacer groups include long-chain diamines, such ashexamethylene diamine.

Immobilization of an enzyme substrate to a support can also occurelectrostatically (i.e., ionically), although covalent attachment ispreferred. For example, immobilization can occur through the interactionbetween the negatively charged sulphonate groups of an enzyme substrate(e.g., one derivitized with 1-hydroxypyrene-3,6,8-trisulphonate) andpositively charged surface ammonium groups of an anion exchanger used asa solid support.

Particularly preferred reactive supports useful in the present inventionare supports having azlactone-functional groups on internal and/orexternal surfaces of such supports. Such reactive groups have anazlactone-functional group of the following formula:

wherein R₁ and R₂ are independently a (C1-C14) alkyl group, a (C3-C14)cycloalkyl group, a (C5-C12) aryl group, a (C6-C26) arenyl groupoptionally having up to three S, N, and nonperoxidic O heteroatoms, orR₁ and R₂ taken together with the carbon to which they are joined canform a carbocyclic ring containing 4-12 ring atoms, and n=0 or 1.

Azlactone-functional reactive supports are particularly preferredbecause they are generally stable. They also rapidly and directlycovalently couple enzyme substrates, optionally with spacer groups,better and with fewer side reactions (e.g., hydrolysis) than othersupports having reactive functional groups. Furthermore, they possesshigh covalent coupling capacities with nucleophiles.Azlactone-functional reactive supports can be made by a number ofmethods as disclosed in U.S. Pat. No. 5,561,097 (Gleason et al.).

Enzyme Substrates

A wide variety of enzyme substrates (ES) can be used. Preferably, theenzyme substrates include a group capable of interacting with, or morepreferably reacting with, the hydroxyl, amino, or sulfhydryl moiety, forexample, of a solid support such that the reaction results in thebonding of the ES to the support. Preferably, the mechanism of bondingto the support does not interfere with the ability of the enzyme to acton the ES or destroy the ability of the ES to produce a signal whenacted upon by the enzyme.

Enzyme substrates include those that interact with enzymes to give adetectable signal. Examples include, but are not limited to, enzymesubstrates for beta-galactosidase (beta-gal), beta-glucuronidase,alcohol dehydrogenase or other NAD oxidoreductases, transferases,alkaline phosphatases or other hydrolases, lyases, isomerases, oxidases,gyrases, nucleases (DNases and RNases), and restriction enzymes. Suchenzymes are produced by bacteria. Enzyme substrates typically can bepolypeptides, carbohydrates (e.g., polysaccharides), or fatty acidderivatives.

A suitable enzyme substrate preferably includes a detectable label.Examples of such labels include fluorescent, luminescent, andchromogenic labels. Preferred labels are fluorescent. Examples offluorescent labels include coumarin, fluorescein and fluoresceinderivatives. Examples of lumiescent labels include adamantyl oxiranederivatives. Examples of chromogenic labels include sulphonphthaleins,sulphonphthalein derivatives, and indoxyl compounds and theirderivatives.

Specific examples of enzyme substrates include coumarin-4-acetic acid7-O-caprylate, coumarin-4-acetic acid 7-O-beta-D-glucuronide, andcoumarin-4-acetic acid 7-O-beta-D-galactopyranoside.

The immobilization of coumarin-4-acetic acid7-O-beta-D-galactopyranoside via an azlactone-functionalized solidsupport has the following structure (wherein “B-D- refers to “beta-D”):

The immobilized dye resulting from this process is a synthetic reactantfor dealkylases (such as galactosidase). Such enzymes will split theether groups, thereby producing a signal from the immobilizedfluorescent dye 7-hydroxy-coumarin-4-acetic acid (i.e., a derivative ofthe acetic acid- functional dye), which may be detected.

Not only synthetic enzyme substrates, but also naturally occurringenzyme substrates labeled with a dye can be immobilized on a solidsupport. For example, egg albumin lysozyme can be immobilized on a solidsupport and then labeled with a fluorescent dye, e.g.,fluoresceinisothiocyanate. Upon contact with the enzyme trypsin thisenzyme substrate will decompose releasing fluorescein andfluorescein-marked fragments of the lysozyme.

Bacteria and Bacteriophages

The type of bacteria that can be detected using the method andimmobilized enzyme substrate of the present invention is not limited.Suitable target bacteria that can be hosts for bacteriophage anddetectable according to the present invention include, but are notlimited to, those of the following genera: Escherichia, Enterobacter,Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Klebsiella,Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma,Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium,Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia,Chromobacterium, Brucella, Yersinia, Haemophilus, and Bordetella. Suchbacteria can also be used as reagents in methods of the presentinvention.

The type of bacteriophage that can be detected using the method andimmobilized enzyme substrate of the present invention is not limited.Suitable target bacteriophage that can interact with host bacteria andbe detectable according to the present invention include, but are notlimited to, the following: Escherichia phage, Enterobacter phage,Salmonella phage, Staphylococcus phage, Shigella phage, Listeria phage,Aerobacter phage, Klebsiella phage, Proteus phage, Pseudomonas phage,Streptococcus phage, Chlamydia phage, Mycoplasma phage, Pneumococcusphage, Neisseria phage, Clostridium phage, Bacillus phage,Corynebacterium phage, Mycobacterium phage, Campylobacter phage, Vibriophage, Serratia phage, Providencia phage, Chromobacterium phage,Brucella phage, Yersinia phage, Haemophilus phage, and Bordetella phage.Such phage can also be used as reagents in methods of the presentinvention. They are typically available from the American Type CultureCollection (ATCC) or can be isolated from nature, and can be used in theform of lyophilized pellets, for example.

A wide variety of enzymes are produced upon the lysis of bacterial cellsusing bacteriophage. The released enzyme reacts with an immobilizedenzyme substrate to give a detectable signal. Examples include, but arenot limited to, beta-galactosidase (beta-gal), beta-glucuronidase,alcohol dehydrogenase or other NAD oxidoreductases, transferases,alkaline phosphatases or other hydrolases, lyases, isomerases, oxidases,gyrases, nucleases (DNases and RNases), and restriction enzymes.

Conditions effective for such lysis to occur are generally well known toone skilled in the art. Such conditions are disclosed in E. L. Ellis etal., “The growth of bacteriophage,” J. Gen. Physiol., 22, 365 (1939),and typically include sufficient time at a temperature of about 37° C.under conditions suitable for bacterial growth.

Types of Assay

The method and immobilized enzyme substrate of the present invention canbe used in a variety of assays. The immobilized enzyme substrate can beadded with bacteriophage to a sample of interest for detecting a targetbacteria. The bacteriophage chosen is one that will infect andsubsequently lyse the bacteria of interest. Alternatively, theimmobilized enzyme substrate can be added with bacteria to a sample ofinterest for detecting a target bacteriophage. The bacteria chosen isone that is susceptible to (i.e., can be infected and lysed by) thebacteriophage of interest.

In one embodiment, a method of detecting a target bacteriophageincludes: combining bacteria and a sample of interest to form a reactionmixture; incubating the reaction mixture under conditions effective forany target bacteriophage present in the sample of interest to lyse thebacteria and release enzyme; adding an immobilized enzyme substrate tothe reaction mixture; and monitoring the reaction mixture for adetectable signal produced from interaction between the immobilizedenzyme substrate and any released enzyme present. Adding the immobilizedenzyme substrate to the reaction mixture can occur before or afterincubating the reaction mixture.

In another embodiment, a method of detecting target bacteria includes:combining bacteriophage and a sample of interest to form a reactionmixture; incubating the reaction mixture under conditions effective forthe bacteriophage to lyse any target bacteria present in the sample ofinterest and release enzyme; adding an immobilized enzyme substrate tothe reaction mixture; and monitoring the reaction mixture for adetectable signal produced from interaction between the immobilizedenzyme substrate and any released enzyme present. Adding the immobilizedenzyme substrate to the reaction mixture can occur before or afterincubating the reaction mixture.

These methods can involve a qualitative or quantitative determination.For a quantitative determination of bacteriophage, for example, thereaction mixture can be plated out on an appropriate growth medium, andthe areas emitting the detectable signal are counted. Such areastypically become plaques (i.e., areas of clearing orbacteriophage-derived discontinuity on a lawn of bacterial “helpercells”) with sufficient time. A standard test method for plaquedetection is described in Standard Test Method for Coliphages in Water,ASTM Designation: D4201-82 (Reapproved 1989).

Preferably, a quantitative assay for bacteria involves phageamplification to detect bacteria by observing the formation of plaques.Generally, this method involves: combining bacteriophage and a sample ofinterest to form a reaction mixture; allowing the bacteriophage toinfect any target bacteria present in the sample of interest; adding anantiviral agent to inactivate any extracellular bacteriophage; addingbacterial helper cells to the reaction mixture; adding an immobilizedenzyme substrate to the reaction mixture; incubating the reactionmixture under conditions effective for the bacteriophage to lyse anytarget bacterium present and the bacterial helper cells and releaseenzyme; and monitoring the reaction mixture for a detectable signalproduced from interaction between the immobilized enzyme substrate andany released enzyme present. If no target bacteria are present, thebacteriophage will be all inactivated by the antiviral agent and nolysing of the bacterial helper cells will occur. This method ispreferably used for the quantitative determination of bacteria in asample, although it can also involve a qualitative determination.

The immobilized ES would react with the bacterial enzyme released bybacteriophage infection of the target bacteria and subsequent breaking(lysis) of the cell wall, but the immobilized ES would not cross abacteria cell wall to react with enzyme still within intact bacteriacells. Therefore, an immobilized ES could be utilized in the finalstages of an assay to produce a signal indicating the presence of targetbacteria in the original sample with little or no interference frombacteria “helper cells” or non-target bacteria.

Suitable bacterial helper cells can be the same or different than thetarget bacteria. Preferably, they are closely related to the targetbacteria such that they can be infected by the chosen bacteriophage.Examples of bacterial helper cells include those listed above for thetarget and reagent bacteria.

The phage amplification can occur on a wide variety of culture mediaknown to one of skill in the art. Typically, a culture media includesvarious nutrients, including a carbon source such as a carbohydrate, anda nitrogen source such as an amino acid or protein hydrolysate.Alternatively, the amplification can occur on a solid or semi-solidculture device such as a “PETRIFILM” device as disclosed in U.S. Pat.No. 5,958,675 (Wicks et al.)

Also, the method and immobilized enzyme substrate can be incorporatedinto the device disclosed in U.S. patent application Ser. No. 09/434,586(Wicks et al.) filed on Nov. 5, 1999, which includes at least twochambers separated by an activatable seal (i.e., a component, such as avalve, that separates two compartments so as to prevent leakage) whereinupon activation of the seal, the two chambers are in communication.Preferably, this device is in the form of a tube, which can have avariety of cross-sectional shapes, although other constructions (e.g.,rectangular or circular tubes, channels on a flat substrate, ormicroreplicated structures) are envisioned.

In a preferred embodiment, the device is used in the detection ofbacteria (target bacteria) by adding bacteriophage to a test sample toinfect the target bacteria in the test sample, killing the extracellularbacteriophage with an antiviral (or mixture of antivirals, such asferrous salts, cuprous salts, leaf extracts, pomegranate rind extracts,and organic acids, suitable for killing extracellular bacteriophage),neutralizing the antiviral (for example, with a buffer), adding animmobilized ES, and amplifying the bacteriophage by incubating theresulting mixture in the presence of a lawn of bacterial helper cells.Such phage amplification assays that use plaque formation as theend-point (and no ES) are known to those of skill in the art and aredisclosed in U.S. Pat. No. 5,498,525 (Rees et al.). In the presence ofan immobilized ES, such as described in Example 4 of the presentinvention, the appearance of a fluorescent signal is an indication thatthe test sample contained the target bacteria. The assay results in theform of a fluorescent signal can be read rapidly, typically within aboutfour hours to about six hours, and confirming at 24 hours, if needed.Conventional methods to enumerate bacteria usually require about 24hours to about 48 hours of growth.

EXAMPLES

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention. Unless otherwiseindicated, all parts and percentages are by weight.

Example 1

Enzyme Substrate Bonded to an Insoluble Support

The objective of this experiment was to prepare an enzyme substrate(coumarin-4-acetic acid 7-O-beta-D-galactopyranoside) covalently bondedto an insoluble support substance (azlactone beads).

A sample (10 g) of azlactone beads (Pierce Chemical Co., Rockford, Ill.)was derivatized with 1,6-hexanediamine (as described in Example 11 ofU.S. Pat. No. 5,561,097) to provide a free amino group at each azlactonesite. Approximately 41 mmole amine equivalents were used per ml ofazlactone beads suspension. A sample (200 mg) of the derivatizedazlactone beads was added to a solution of the fluorogenic substratecoumarin-4-acetic acid 7-O-beta-D-galactopyranoside (10 mg, Sigma, St.Louis, Mo.), dicyclohexyl carbodiimide (DCC) (5.4 mg, Aldrich Chemical,Milwaukee, Wis.), and 1-hydroxybenzotriazole (HOBt) (7 mg, AldrichChemical) all in DMF to provide a total volume of 3 ml. The resultingmixture was constantly agitated in a laboratory tube rocker for fivedays at room temperature. The supernatant liquid was discarded and thebeads were washed with copious amounts of water and were resuspended inwater (1 ml). The resulting suspension showed very little fluorescenceand, after the suspension was centrifuged, all of the fluorescence wascarried by the bead pellet. The supernatant liquid from thecentrifugation did not give a fluorescent signal over background whenthe purified enzyme beta-galactosidase (beta-gal) was added. However,when beta-gal was added to the bead pellet resuspended in fresh buffer(Butterfield's buffer, Fisher Scientific, Pittsburgh, Pa.), a verystrong fluorescent signal was observed.

Example 2

Enzyme Substrate Bonded to an Insoluble Support in the Presence of E.Coli Cells and Bacteriophage

The objective of this experiment was to observe the fluorescent signalproduced from an enzyme substrate attached to an insoluble support inthe presence of E. coli cells, both in the presence and absence ofbacteriophage.

A sample (50 mg of beads) of the enzyme substrate coumarin-4-acetic acid7-O- beta-D-galactopyranoside covalently bonded to the insolubleazlactone beads support (as described in Example 1) was added toButterfield's buffer (1 ml, pH 7, Fisher Scientific) that contained E.coli 625 cells (Silliker Laboratories, Chicago Heights, Ill. having aninitial concentration of approximately 10⁸ cells/ml. The resultingmixture was incubated for 6-10 hours at 37° C. both in the presence andabsence of bacteriophage RB33s (10⁶ phage, T4 Laboratory, EvergreenState College, Olympia, Wash.) that was capable of lysing the E. colicells. A sample of the substrate-beads in Butterfield's buffer mediumonly was also incubated in the same manner and showed little or nofluorescent signal. The incubated medium containing E. coli cells but nobacteriophage showed only a low background level of fluorescence. Theincubated medium containing the E. coli cells and the bacteriophageshowed a higher level of fluorescence. This higher level of fluorescencewas attributed to the lysis of E. coli cells by the bacteriophage andthe subsequent release of the residual beta-gal enzyme. The enzyme wasthen able to catalyze the hydrolysis of the beads-supported enzymesubstrate to produce a hydrolysis product that emitted a highlyfluorescent signal.

Example 3

Comparison of Bonded Substrate to Non-Bonded (Free) Substrate in thePresence of E. Coli Cells

The objective of this experiment was to show that an enzyme substrate(coumarin-4-acetic acid 7-O-beta-D-galactopyranoside) covalently bondedto an insoluble support substance (azlactone beads) produces little orlow levels of fluorescent signal relative to free coumarin-4-acetic acid7-O-beta-D-galactopyranoside in solution.

A sample (about 200 mg of beads) of the enzyme substratecoumarin-4-acetic acid 7-O-beta-D-galactopyranoside covalently bonded tothe insoluble azlactone beads support (as described in Example 1) wassuspended in 500 μl of sterile water and then 100 μl of this suspensionwas added to each of 5 wells of a 96-well plate (Co-Star, V.W.R.,Chicago, Ill.). The quantity of beads was chosen so that hydrolysis ofthe enzyme substrate would be expected to give a fluorescent signal ofabout 4000 relative fluorescent units (RFU). Other wells were filledwith free coumarin-4-acetic acid 7-O-beta-D-galactopyranoside (50 μg/ml)and contained no beads. A 100-μl aliquot of either E. coli 650 or E.coli 651 cells (prepared by centrifuging 1 ml of an overnight culture ofeach bacterium, washing 2× with Butterfield's buffer, and resuspendingin 500 μl of fresh Luria-Bertani (LB) Broth) was then added to thesample wells. The well plates were then incubated at 37° C. for up to 6hours. Changes in the fluorescence of each well were determined using aCambridge Instruments Model 7620 Fluorescent Microplate Reader(Cambridge Instruments, Boston, Mass.). Those wells containing beadswith covalently bound enzyme substrate gave little or no fluorescencewhile those that contained the free enzyme substrate in solution showeda marked increase in fluorescent signal (see Table1).

TABLE 1 Fluorescence (RFU)¹ at Indicated Time E. Coli (Hours) SampleStrain 0 1.5 2.5 4 Free Enzyme 560 161 226 337 717 Substrate 561 156 300436 592 Bonded 560 155 153 157 166 Enzyme Substrate 561 148 170 160 175¹Each data point represents the average of 5 replicates.

Example 4

Detection of Bacteria Utilizing an Immobilized Enzyme Substrate

A phage amplification device (PAD) having separate chambers A, B, C, andD is constructed as described in Example 3 of U.S. patent applicationSer. No. 09/434,586 (Wicks et al.). Center chambers B and C containantiviral components. Following construction, a pellet of lyophilized E.coli, ATCC 13706 bacteria (approximately 1×10⁸ cfu/ml) is added toChamber D to serve as bacteria “helper cells”. Additionally, a sample(50 mg of beads) of the enzyme substrate coumarin-4-acetic acid7-O-beta-D-galactopyranoside covalently bonded to the insolubleazlactone beads support (as described in Example 1) is added to ChamberD. Chamber A is left empty to receive the test sample and all valves areset initially in a “closed position.”

An overnight culture of E. coli, ATCC 13706 containing 1×10⁸ cfu/ml isdiluted ten-fold stepwise in Lambda buffer (as described in Example 4 ofU.S. Pat. No. 5,498,525 (Rees et al.)) so that Chamber A of the PADcontains approximately 0.1 ml of culture solution. To this sample isadded 10 μl of a Nutrient Broth (Product No. 4311479, BBL, Cockysville,Md.) suspension of bacteriophage [Phi×174 (ATCC 13706-B1)] containing1×10¹¹ pfu/ml. The bacteriophage is allowed to adsorb to the bacteriacells for 10 minutes at 37° C. in an incubator. Valve 1 (betweenChambers B and C) is then opened and the antiviral components ofChambers B and C are allowed to mix for 2 minutes at 23° C. Non-adsorbedbacteriophage is then inactivated by opening Valve 2 (between Chambers Aand B) and allowing the antiviral solution to mix with the contents ofChamber A for 5 minutes at 23° C. The resulting solution is thenneutralized by opening Valve 3 (between Chambers C and D) and combiningthe solution with the bacteria “helper cells” pellet and immobilizedenzyme substrate in Chamber D for 5 minutes at 23° C. The PAD is thenincubated at 23° C. for up to 6 hours and changes in fluorescence aredetermined. The fluorescent signal is an indication of E. coli bacteriain the original culture.

The complete disclosures of the patents, patent documents, andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousmodifications and alterations to this invention will become apparent tothose skilled in the art without departing from the scope and spirit ofthis invention. It should be understood that this invention is notintended to be unduly limited by the illustrative embodiments andexamples set forth herein and that such examples and embodiments arepresented by way of example only with the scope of the inventionintended to be limited only by the claims set forth herein as follows.

What is claimed is:
 1. A method of detecting target bacteria, the methodcomprising: combining bacteriophage and a sample to form a reactionmixture; incubating the reaction mixture under conditions effective forthe bacteriophage to lyse any target bacteria present in the sample,thereby releasing enzyme; providing an immobilized enzyme substrate inwhich an enzyme substrate is covalently coupled to a solid supportthrough a reactive functional group on the support; adding theimmobilized enzyme substrate to the reaction mixture so that at least aportion of the immobilized enzyme substrate reacts with enzyme releasedby the lysed target bacteria but does riot react with enzyme withinintact bacteria; permitting enzyme released from lysed target bacteriato react with the immobilized enzyme substrate, thereby producing adetectable signal; and detecting the detectable signal.
 2. The method ofclaim 1 wherein adding an immobilized enzyme substrate to the reactionmixture occurs prior to incubating the reaction mixture.
 3. The methodof claim 1 wherein detecting the detectable signal comprisesquantitatively determining the amount of target bacteria present in thesample.
 4. The method of claim 1 wherein the enzyme substrate iscoumarin-4-acetic acid 7-O-caprylate, coumarin-4-acetic acid7-O-beta-D-glucuronide, or coumarin-4-acetic acid7-O-beta-D-galactopyranoside.
 5. The method of claim 1 wherein thebacteriophage are Escherichia phage, Enterobacter phage, Salmonellaphage, Staphylococcus phage, Shigella phage, Listeria phage, Aerobacterphage, Klebsiella phage, Proteus phage, Pseudomonas phage, Streptococcusphage, Chlamydia phage, Mycoplasma phage, Pneumococcus phage, Neisseriaphage, Clostridium phage, Bacillus phage, Corynebacterium phage,Mycobacterium phage, Campylobacter phage, Vibrio phage, Serratia phage,Providencia phage, Chromobacterium phage, Brucella phage, Yersiniaphage, Haemophilus phage, or Bordetella phage.
 6. The method of claim 1wherein the solid support comprises an azlactone-functional solidsupport.
 7. The method of claim 1 wherein the detectable signal is afluorescent signal, luminescent signal, or chromogenic signal.
 8. Themethod of claim 7 wherein the detectable signal is a fluorescent signal.9. The method of claim 1 wherein the solid support comprises one or moreazlactone-functional groups.
 10. A method of detecting target bacteria,the method comprising: combining bacteriophage and a sample to form areaction mixture; allowing the bacteriophage to infect any targetbacteria present in the sample; adding an antiviral agent to inactivateany extracellular bacteriophage; adding bacterial helper cells that arecapable of being infected by the bacteriophage to the reaction mixture;providing an immobilized enzyme substrate in which an enzyme substrateis covalently coupled to a solid support through a reactive functionalgroup on the support; adding the immobilized enzyme substrate to thereaction mixture so that at least a portion of the immobilized enzymesubstrate reacts with enzyme released by the lysed target bacteria butdoes not react with enzyme within intact bacteria; incubating thereaction mixture under conditions effective for the bacteriophage tolyse any target bacteria present and the bacterial helper cells, therebyreleasing enzyme; permitting enzyme released from lysed target bacteriaand the lysed bacterial helper cells to react with the immobilizedenzyme substrate, thereby producing a detectable signal; and detectingthe detectable signal.
 11. The method of claim 10 wherein detecting adetectable signal comprises quantitatively determining the amount oftarget bacteria present in the sample.
 12. The method of claim 11wherein quantitatively determining the amount of target bacteria presentin the sample comprises plating the reaction mixture of the bacterialhelper cells, immobilized enzyme substrate, and sample of interest on agrowth medium, and counting areas emitting the detectable signal. 13.The method of claim 10 wherein the solid support comprises one or moreazlactone-functional groups.